Background: High-performance liquid chromatography (HPLC) has emerged as a highly sensitive and versatile analytical technique for quantifying antihypertensive drugs, such as Captopril (CAP). This study focused on the optimization and validation of an HPLC method for quantifying CAP in an in vitro hydrogel permeability test. Objectives: The main objective of this study was to develop and validate an HPLC method for quantifying CAP in an in vitro hydrogel permeability test. Methods: The HPLC method employed a C18 column (Waters, Sunfire, 5 μm; 250 × 4.6 mm) and a mobile phase consisting of methanol-water (85% v/v orthophosphoric acid) in a 55:45 (v/v) ratio at a flow rate of 0.5 mL/min. The UV-Vis detector was configured to detect CAP at a wavelength of 220 nm. The hydrogel film used in the permeability test was prepared using poly (vinyl alcohol)/poly (vinyl caprolactam) (PVA/PNVCL) with citric acid as a crosslinking agent. Results: All results met the validation parameters according to ICH Guideline. The HPLC method showed consistent retention time (4.7–4.9 min), linearity (1–50 µg/mL; r = 0.9995), accuracy (98.11-101.78%), precision (RSD ≤ 2%), and LoD/LoQ (0.19/0.62 µg/mL). The developed HPLC method was successfully applied to an in vitro permeability test using horizontal diffusion cells. The results demonstrated that CAP permeated through the swollen hydrogel film, with a cumulative drug permeation exceeding 30%. Conclusion: This highlighted the method’s utility in assessing drug transport properties through hydrogels. The validated HPLC method demonstrates robustness and reliability for quantifying CAP in the hydrogel permeability test. © The Author(s), under exclusive licence to Tehran University of Medical Sciences 2024.